The present invention relates to gene therapy. In particular, therapeutic agents and methods useful in targeting and delivering genes more efficiently to particular cells are disclosed, wherein re-targeted, tropism-modified viral vectors presenting ligand on the surface and including a nucleotide sequence encoding a therapeutic gene product are used to facilitate targeting and delivery.
The primary impediment to the transfer of non-native or foreign DNA into mammalian cells is that the genetic material must be transported across multiple cellular barriers before it enters the host cell nucleus and initiates gene expression. Previously established methods have utilized artificial means to introduce DNA into the cell although these methods are associated with significant cell toxicity (Graham et al., Virology 52:456-467 (1973); Felgner et al, PNAS USA 84:7413-7417 (1987)).
More recently, enhanced transfer of DNA conjugates into cells has been achieved with adenovirus, a human DNA virus which readily infects various cell types (Horwitz, Adenoviridae and their replication, in Virology, Fields and Knipe, eds., Raven Press, NY (1990) pp. 1679-1740). Since adenovirus efficiently disrupts the membranes of endocytic vesicles, co-internalization of the virus with the DNA conjugate allows rapid transfer of the conjugate into the cell cytoplasm before it can be subjected to lysosomal degradation. The fact that adenovirus exhibits selective tropism has also been exploited to reconstitute these cells in vivo with the human cystic fibrosis transmembrane conductance regulator (CFTR) (Rosenfeld et at., Cell 68:143-155 (1992)) and the alpha 1-antitrypsin genes (Rosenfeld et al., Science 252:431-434 (1991)).
A number of features make adenoviruses very attractive for gene delivery applications. Knowledge of the adenovirus genetic system is fairly extensive, including the viral life cycle, DNA replication, transcription and RNA processing, and regulation of virus gene expression. In addition, the size of the adenovirus (Ad) genome allows relatively easy manipulation of the viral DNA while still having the capacity for insertion of most cDNAs into the viral genome. Additional advantages of adenovirus vectors include their ability to infect both dividing and nondividing cells efficiently, to induce high-level foreign protein expression without replication or integration of the viral genome, and to grow to high yields when propagated in an appropriate complementing cell line.
If a target tissue lacks sufficient levels of adenovirus attachment receptors to mediate viral adsorption, however, this may also be a barrier to efficient gene transfer. Infection by most viruses requires viral attachment to its host cell receptor. Adenovirus attaches to its host cell receptor via its fiber protein (see, e.g., Wickham et al., Cell 73:309-319 (1993)).
The Ad fiber protein is a long, trimeric protein that protrudes from the surface of the virion. At the distal end of the fiber protein is a knob-like C-terminus that interacts with an unidentified cellular receptor present on HeLa and other epithelial-derived carcinoma cell lines (see, e.g., Defer et al., J. Virol. 64:3661-3673 (1990)). The receptor, generally identified as FibR, is assumed to be expressed by cells that are the normal targets for adenovirus infection.
Thus, reduced gene delivery to certain tissues may well result from a low expression of the adenovirus receptor (FibR). A lack of functional receptors is thus likely to be directly correlated with dramatic reductions in gene transfer efficiency.
In general, adenoviral vectors possess the capacity for in vivo gene transfer that are critical to effective gene therapy. Following administration of the adenovirus vector, three distinct, sequential steps are required for expression of the therapeutic gene in target cells: (1) attachment of the adenovirus vector to specific receptors on the surface of the target cell; (2) internalization of the virus; and (3) transfer of the gene to the nucleus where it can be expressed. Thus, any attempt to modify the tropism of an adenovirus vectorxe2x80x94that is, its native ability to target its cognate receptor must reserve its ability to perform these three functions efficiently.
Investigators have met with greater or lesser success in this regard. For example, the methodology proposed by Krasnykh et al. (J. Virol. 70: 6839-46 (1996) generates recombinant adenovirus with chimeric fibers having a fiber shaft from one Ad serotype and a knob from another, thereby altering the adenovirus"" receptor recognition profile. (Also see Gall et al., J. Virol. 70:2116-2123 (1996), which describes an Ad 5/7 capsid chimera.) However, such constructs would appear to have limited utility, as they still rely on the less-than-ubiquitous (and less-than-efficient) Ad receptors for targeting. Moreover, Ad vectors that rely upon Ad receptors for targeting (and putative internalization) are not able to target as wide a variety of cells as one might wish, and depending on the nature of the chimeric fiber, any alterations in its conformation may have a negative impact on targeting and/or delivery.
Further, the modifications described in the aforementioned articles do not alter viral tropism in a manner that enhances viral targeting or increases trafficking to the nucleus, contrary to what is disclosed herein. In addition, the art fails to disclose targeting and delivery constructs and systems that achieve the unexpectedly high level of xe2x80x9cinfectivityxe2x80x9d and expression shown herein. Finally, the constructs and methods of the present invention successfully achieved delivery of therapeutic agents to cells that are normally resistant to viral-mediated delivery.
In view of the aforementioned problems, the design and construction of the within-disclosed vectors and conjugates provides a novel and elegant solution, as described further herein. The use of the recombinant sequences and vectors of this invention to mediate the transfer of foreign genes into recipient cells both in vitro and in vivo overcomes the limitations of the above-described gene transfer systems. This invention utilizes recombinant constructs which confer the advantages of targeting via the fibroblast growth factor receptor upon adenovirusxe2x80x94in place of the adenovirus usual targeting via fiber proteinxe2x80x94and thus represents an improved method for gene therapy as well as for therapeutic applications involving delivery of a gene.
In contrast to the disadvantages of using intact adenovirus, modified adenovirus vectors requiring a helper plasmid or virus, or so-called replication-deficient adenovirus, the use of recombinant adenovirus-derived vectors according to the present invention provides certain advantages for gene delivery. In particular, adenoviral vectors having their native tropism modified or ablated, which are then re-targeted via a targeting ligand, are disclosed herein as advantageous for a variety of gene therapy applications.
The Ad-derived vectors of the present invention possesses all of the functional properties required for gene therapy including binding to specific cell receptors and penetration of endocytic vesicles. They further include those in which all or part of the fiber head or tail is replaced withxe2x80x94or conjugated toxe2x80x94a ligand-encoding gene. Use of the vectors and conjugates disclosed herein allows one to target a wide variety of cells and to deliver therapeutic agentsxe2x80x94irrespective of whether those agents are proteins, polypeptides, nucleotide sequences, or some other molecular speciesxe2x80x94directly into specific target cells.
The presently-disclosed constructs, systems and methods afford a level of flexibility in therapeutic approaches not seen with other systems and methods. Therefore, the vectors, systems and methods of the present invention are ideal for use in a wide variety of therapeutic applications.
Therefore, in one embodiment, the present invention provides a tropism-modified adenoviral vector system that specifically targets cells expressing a preselected receptor, comprising an antibody or fragment thereof that binds an adenoviral capsid protein; a targeting ligand that binds the preselected receptor; and an adenovirus containing a nucleic acid molecule that encodes a therapeutic gene product under the control of a promoter; wherein the ligand is conjugated to the antibody or fragment thereof and wherein the antibody or fragment thereof is bound to the adenovirus. In one variation, the ligand is conjugated to the antibody or fragment thereof as a fusion, e.g., a fusion-sFv. In another variation, the promoter is a tissue-specific promoter.
In another embodiment, a tropism-modified adenoviral vector is provided wherein the targeting ligand is a polypeptide reactive with an FGF receptor. In one variation, the polypeptide reactive with an FGF receptor is an antibody or fragment thereof. In another variation, the antibody is a single-chain antibody. In one alternative embodiment, the antibody is 11A8. In another, the polypeptide reactive with an FGF receptor is selected from the group consisting of FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-11, FGF-13, FGF-14, FGF-15, and molecules having 20% or greater homology to any of the foregoing. In still another embodiment, the polypeptide reactive with an FGF receptor is FGF-2. Yet another variation provides that the targeting ligand is selected from the group consisting of a polypeptide reactive with a VEGF receptor, a polypeptide reactive with a PDGF receptor, and a polypeptide reactive with an EGF receptor.
In other variations of the disclosed invention, the surface-presented ligand is a polypeptide reactive with a cell-surface receptor; growth factor receptors are one class of receptors contemplated within the scope of the present invention. Another class of receptors contemplated within the scope of the invention includes receptors for Her-2/neu or erbB2. Thus, a polypeptide reactive with a cell-surface receptor according to the present invention includes antibodies or fragments thereof, including single-chain antibodies, which react with receptors for Her-2/neu or erbB2.
The present invention also discloses embodiments wherein the native tropism of the viral vector is modified; in still other embodiments, the native tropism of the viral vector is ablated. In various preferred embodiments, the vector is an adenoviral vector. The adenoviral vector is readily selected from any of the adenovirus serotypes, as well.
In a further aspect of the present invention, a tropism-modified vector is disclosed wherein the therapeutic gene product is a cytocide or a prodrug. In one set of related embodiments, the cytocide is a ribosome inactivating protein. In other variations, the gene product is thymidine kinase, cytosine deaminase, or nitroreductase.
According to various embodiments of the present invention, the therapeutic gene product enhances cellular proliferation. In one variation, the therapeutic gene product is a biologically active protein or polypeptide that augments or complements an endogenous protein. In another variation, the therapeutic gene product enhances cellular differentiation. In still another variation, the therapeutic gene product is a molecule which enhances tissue repair or regeneration. Yet another variation provides that the therapeutic gene product is a molecule which stimulates a protective immune response.
The present invention further discloses a variety of pharmaceutical compositions. In one embodiment, a pharmaceutical composition of the present invention comprises a physiologically acceptable buffer and a tropism-modified adenoviral vector presenting a ligand on its surface, wherein the vector includes a nucleic acid molecule encoding a therapeutic gene product under the control of a promoter. In one variation, the ligand is a polypeptide reactive with an FGF receptor. In various alternative embodiments, the polypeptide reactive with an FGF receptor is selected from the group consisting of FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-11, FGF-13, FGF-14, and FGF-15. In one preferred variation, the polypeptide reactive with an FGF receptor is FGF-2. In other embodiments, the polypeptide reactive with an FGF receptor is an antibody or fragment thereof. In one alternative variation, the antibody is a single-chain antibody. In another variation, the antibody is 11A8.
In other variations of the disclosed invention, the surface-presented ligand is a polypeptide reactive with a cell-surface receptor; growth factor receptors are one class of receptors contemplated within the scope of the present invention. Another class of receptors contemplated within the scope of the invention includes receptors for Her-2/neu or erbB2. Thus, a polypeptide reactive with a cell-surface receptor according to the present invention includes antibodies or fragments thereof, including single-chain antibodies, which react with receptors for Her-2/neu or erbB2.
In various aspects of the present invention, the ligand is genetically fused with an adenoviral capsid protein. In others, the ligand is chemically conjugated to an adenoviral capsid protein. In one variation, the ligand is conjugated to an antibody or fragment thereof that binds a viral capsid protein. In another variation, the ligand is conjugated to the antibody or fragment thereof as a fusion, e.g., a fusion-sc-Fv.
Other variations contemplate that the therapeutic gene product is selected from the group consisting of protein, ribozyme and antisense. In one alternative embodiment, the therapeutic gene product is a cytocide. Exemplary cytocides include ribosome-inactivating proteins. In another embodiment, the therapeutic gene product is a prodrug. Exemplary prodrugs include thymidine kinase, cytosine deaminase, or nitroreductase. Other embodiments disclose a wide variety of therapeutic gene products, including products that replace or repair defective, improperly regulated, or nonfunctional genes. In various alternative embodiments, therapeutic gene products within the context of the present invention stimulate wound healing, tissue repair, connective tissue growth, angiogenesis, or the amelioration of ischemia, to name a few examples. In other embodiments, therapeutic gene products treat, interfere with or block a disease process, such as hyperproliferation of cells, tumor growth, metastasis, and the like.
Thus, the present invention also discloses a variety of treatment methods. In one embodiment, the invention contemplates a method of treating tumors, comprising administering a pharmaceutical composition comprising a physiologically acceptable buffer and a tropism-modified adenoviral vector presenting a ligand on its surface, wherein the vector includes a nucleotide sequence encoding a therapeutic gene product under the control of a promoter, wherein the therapeutic gene product is selected from the group consisting of E-cadherin, BGP, Rb, p53, CDKN2/P16/MTS1, PTEN/MMAC1, APC, p33ING1, Smad4, maspin, VHL, WT1, Men1, NF2, MXI1, and FHIT. The invention also provides methods of treating ischemia, comprising administering a pharmaceutical composition comprising a physiologically acceptable buffer and a tropism-modified adenoviral vector presenting a ligand on its surface, wherein the vector includes a nucleotide sequence encoding a therapeutic gene product under the control of a promoter, wherein the therapeutic gene product is selected from the group consisting of IGF, TGFxcex21, TGFxcex22, TGFxcex23, HGF, VEGF 121, VEGF 165, FGF1, FGF2, FGF4, FGF5, PDGF-A, and PDGF-B.
In still other variations, the invention provides methods of treating connective tissue injury, comprising administering a pharmaceutical composition comprising a physiologically acceptable buffer and a tropism-modified adenoviral vector presenting a ligand on its surface, wherein the vector includes a nucleotide sequence encoding a therapeutic gene product under the control of a promoter, wherein the therapeutic gene product is selected from the group consisting of PTH, BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8, BMP10, BMP11, mammalian BMP, and Xenopus BMP. An alternative method comprises the administration of a pharmaceutical composition comprising a physiologically acceptable buffer and a tropism-modified adenoviral vector presenting a ligand on its surface, wherein the vector includes a nucleotide sequence encoding a therapeutic gene product under the control of a promoter, wherein the therapeutic gene product is selected from the group consisting of Bovine VEGF, VEGF, VEGF-B, VEGF-C, Angiopoietin-1, Angiogenin, IGF-1, IGF-II, HGF, PDGF A, PDGF B, TGFB1, TGFB2, and TGFB3.
The present invention also discloses various methods of treating malignancies, including cancer. In one such embodiment, a method of treating cancer is disclosed, comprising contacting the cancer cells with a pharmaceutical composition comprising a physiologically acceptable buffer and a tropism-modified adenoviral vector presenting a ligand on its surface, wherein the vector includes a nucleotide sequence encoding a therapeutic gene product under the control of a promoter, wherein the therapeutic gene product is selected from the group consisting of HSVTK, VZVTK, nitroreductase, and cytosine deaminase; and contacting the cancer cells with a substrate. In various embodiments, the substrate is a molecule that is acted upon to produce a molecule that is cytotoxic or cytostatic to the cancer cells.
In the various disclosed methods, the ligand is a polypeptide reactive with a specific cellular receptor; various polypeptides useful in this regard are recited hereinabove. In various preferred embodiments, the receptor is an FGF receptor. In one variation, the polypeptide reactive with an FGF receptor is FGF-2. In other variations, the polypeptide reactive with an FGF receptor is selected from the group consisting of FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-11, FGF-13, FGF-14, and FGF-15.
In one alternative embodiment, the ligand is an antibody or a fragment thereof In another embodiment, the antibody is a single-chain antibody. In yet another variation, the ligand is conjugated to an antibody or fragment thereof that binds a viral capsid protein. In various embodiments, the viral capsid protein is adenovirus fiber proteinxe2x80x94for example, an adenovirus knob protein. In yet other embodiments, the ligand may be chemically conjugated to a protein on the surface of a viral vector or may be attached to the capsid as a component of a fusion protein.
In various methods disclosed herein, the therapeutic gene product is selected from the group consisting of protein, ribozyme and antisense. In one alternative embodiment, the therapeutic gene product is a cytocide. Exemplary cytocides include ribosome-inactivating proteins. In another embodiment, the therapeutic gene product is a prodrug. Exemplary prodrugs include thymidine kinase, nitroreductase, and cytosine deaminase. Other embodiments disclose a wide variety of therapeutic gene products, including products that replace or repair defective, improperly regulated, or nonfunctional genes. In various alternative embodiments, therapeutic gene products within the context of the present invention stimulate wound healing, tissue repair, connective tissue growth, angiogenesis, or the amelioration of ischemia, to name a few examples. In other embodiments, therapeutic gene products treat, interfere with or block a disease process, such as hyperproliferation of cells, tumor growth, metastasis, and the like.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various articles and documents are referenced herein to provide further details regarding various procedures, compositions, molecules, and the like. It is expressly to be understood that the disclosures of all publications referred to herein are incorporated by reference in their entirety as though fully set forth herein.